Antibiotic resistance plasmids R6K and RK2 and the mini-F plasmid derivative of Flac are being examined for replication and incompatibility properties in Escherichia coli and other Gram-negative bacteria. The analyses are being carried out at the in vitro and in vivo levels. Using recombinant DNA techniques essential regions for replication and incompatibility of these three plasmid systems have been cloned in E. coli. The activity of these regions in an in vitro replication system anf their nucleotide sequence are being determined. Various low molecular weight derivatives of these plasmids have been generated and their structure altered to produce more effective plasmid cloning vehicles for recombinant DNA studies.